The present invention relates to the quantification of vancomycin in a test sample. In particular, the present invention relates to immunogens, antibodies prepared from such immunogens, and labeled reagents for the specific quantification of vancomycin in a test sample, preferably for use in fluorescence polarization immunoassays.
For the past 30 years, vancomycin has been the drug of choice for the treatment of Gram-positive infections caused by methicillin resistant Staphylococcus aureus. It is also the treatment in bacterial infections in patients allergic to xcex2-lactam antibiotics. Vancomycin is produced by Amycolatopsis orientalis (previously designed Nocardia orientalis and Streptomyces orientalis). Vancomycin is resistant to Gram-negative organisms. Cross resistance with other antibiotics is unknown and in spite of its long usage, there have been few reports of the emergence of resistant organisms during therapy. Vancomycin is not absorbed from the gastrointestinal tract, and hence the antibiotic is used to treat enterocolitis caused especially by Clostridium difficile in the gut. Nagarajan, R., J. Antibiotics, 46:1181 (1993). Vancomycin exerts its antibacterial action by binding preferentially to peptide intermediates involved in the biosynthesis of bacterial cell wall peptidoglycan.
Vancomycin is eliminated via the kidneys. The half life of the drug, 5-11 hours in normal patients, is extended to 2-5 days in patients with renal insufficiency, and is even longer in dialysis patients. While vancomycin is a relatively safe drug; adverse effects which have been observed include nephrotoxicity and autotoxicity.
For safe administration of vancomycin, it is customary to quantify its levels in patient blood. It has been suggested that because the drug stays longer in the body of a renally impaired patient, exposure to internal body temperature for longer periods results in the accumulation of degradation products which are known as Crystalline Degradation Products I and II (CDP-I and CDP-II). CDP-I and CDP-II are rotational isomers which can be separately isolated. Vancomycin and its two major degradation products CDP-I and CDP-II are shown in FIGS. 1-3, respectively.
It is known that vancomycin is unstable in an aqueous environment. U.S. Pat. No. 4,670,258 to Harris, et al. discloses a composition of vancomycin and a tripeptide which is said to stabilize the drug in an aqueous solution. However, such tripeptides can interfere with immunoassay techniques. For example, such interference may occur where an antibody competes with a stabilizing peptide for the same binding site of the analyte.
Historically, vancomycin concentrations in biological fluids have been determined by fluorescence immunoassay (FIA), high performance liquid chromatography (HPLC), radio immunoassay (RIA), the enzyme multiplied immunoassay technique (EMIT) or microbiological techniques. While HPLC is considered by those skilled in the art as the most accurate of all methods for quantification of vancomycin, it is a slow and labor intensive method which requires highly trained personnel and specialized equipment which is not always available in every clinical setting.
More recently, fluorescent polarization techniques have been used to assay for vancomycin. Fluorescent polarization techniques are based on competitive binding immunoassay principles. The principle behind fluorescent polarization is that a fluorescent labeled compound, when excited by a linearly polarized light, will emit fluorescence having a degree of polarization inversely proportional to its rate of rotation. Therefore, when a fluorescent labeled tracer-antibody complex is excited by a linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and emitted. When a xe2x80x9cfreexe2x80x9d tracer compound (i.e., unbound to an antibody) is excited by linearly polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate produced in a competitive binding immunoassay.
Fluorescent polarization techniques and compounds suitable for use as fluorescent labels have been described in the art. For instance, U.S. Pat. Nos. 4,510,251 and 4,614,823, to Kirkemo et al., disclose fluorescent polarization assay for ligands using aminomethyl fluorescein derivatives, respectively. U.S. Pat. No. 4,476,229, to Fino et al., discloses substituted carboxyfluoresceins, including those containing a vancomycin analog, for use in fluorescence polarization immunoassay. U.S. Pat. Nos. 4,420,568 and 5,097,097 to Wang et al., disclose fluorescent polarization immunoassay utilizing substituted triazinylaminofluoresceins as tracers. Wang in U.S. Pat. No. 4,420,568 discloses reaction of vancomycin and dichlorotriazinylaminofluorescein (DTAF). However, this patent does not describe the structure of the product of such a reaction or its application in the heterogeneous system. Griffin et al., (JACS 115, 6482 (1993)) describe a selective method for the synthesis of vancomycin derivatives bearing alkyl, imidazole and amine functional groups attached to the C-terminus and indicated usefulness of this method for preparation of derivatives bearing different functional groups. However, there is no description of the synthesis of immunogenic material or immunocomponents and their use for quantification of vancomycin.
Commercially available fluorescent polarization assays (FPIA) for vancomycin are available. For instance, commercially available assays (Abbott TDX(copyright), TDXFLX(copyright) assays, (hereinafter referred to as the xe2x80x9ccommercially available Abbott Vancomycin assays(s)xe2x80x9d)) include reagents for the quantitative measurement of vancomycin in serum or plasma samples. These assays use a vancomycin derivative labeled with a dichlorotriazinylaminofluorescein (DTAF) (hereinafter referred to as the xe2x80x9ccommercially available tracerxe2x80x9d), and sheep polyclonal antibodies against vancomycin (hereinafter referred to as xe2x80x9ccommercially available antibodiesxe2x80x9d).
FPIAs have an advantage over radioimmunoassays (RIA) in that there are no radioactive substances to dispose of and they are homogeneous assays that can be easily and rapidly performed. However, it has been reported that the commercially available vancomycin assays show an occasional increase in measured vancomycin values which do not conform with HPLC measurements. These increases have been attributed to increased cross-reactivity with CDP-I and CDP-II. As noted above, the isomers CDP-I and CDP-II can be separately isolated. As expected, any solution made from CDP-I will always contain an equilibrium mixture of both isomers. Thus, measures of CDP-I cross-reactivity reported herein measure the cross-reactivity of the equilibrium mixture.
Thus there exists a continuing need for improved assays which can quickly and accurately determine the concentration of vancomycin in the presence of cross-reactive degradation products in biological fluid. Accordingly, the present invention provides unique antibody reagents and labeled reagents for the quantification of vancomycin in a test sample. The invention also provides immunoassay methods which utilize these unique reagents. Also provided are synthetic procedures for preparing immunogens which are employed for the production of such antibody reagents, as well as procedures for preparing such labeled reagents.
Also provided are antibody reagents which can be used, and in many instances are critical, for constructing stable vancomycin calibrators and controls which can be used in assays to measure vancomycin concentration.
According to the present invention, the labeled reagents and the antibody reagents offer an advance in the art beyond previously known procedures when used in an immunoassay for the quantification of vancomycin in a test sample. Specifically, it was discovered that the antibody reagents of the present invention have essentially no cross-reactivity with the metabolites CDP-I and CDP-II. Moreover, the antibody reagents of the present invention can be used in the presence of polypeptides which stabilize the vancomycin molecule for the quantification of vancomycin. In the present invention, the presence of such polypeptides does not interfere with the quantification of vancomycin in a sample.
The present invention provides a method for the quantification of vancomycin in a test sample, wherein:
(a) the test sample is contacted with an antibody reagent having antibodies which are capable of specifically binding to vancomycin and are produced with an immunogen of FIG. 6 wherein P is an immunogenic carrier material, and X is a linking moiety of from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequences cannot contain xe2x80x94Oxe2x80x94O linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms, and a labeled reagent of FIG. 8 wherein Q is a detectable moiety, and X is from 0 to 50 carbon and heteroatoms, including not more than ten heteroatoms, arranged as a straight or branched chain or cyclic moiety, saturated or unsaturated, with the provisos that not more than two heteroatoms may be directly linked in sequence, that the sequence cannot contain xe2x80x94Oxe2x80x94O linkages, that cyclic moieties contain 6 or fewer members, and that branching may occur only on carbon atoms, to form a reaction solution; and
(b) measuring the amount of the labeled reagent in the reaction solution which either is or is not bound with an antibody as a function of the amount of vancomycin in the test sample.
The invention further provides the above method wherein fluorescence polarization is employed.
In a preferred method of the invention the antibody is produced with an immunogen of FIG. 6 and the labeled reagent is as shown in FIG. 8.
The invention further provides novel immunogens of FIG. 6 which are useful to produce antibodies which specifically bind vancomycin.
The invention further provides antibodies which are specific for vancomycin and have essentially no cross-reactivity with CDP I and CDP II and are capable of binding to any non-peptidic site on vancomycin. More specifically, the antibodies of the present invention do not compete with stabilizing peptides, specifically polypeptides used to stabilize vancomycin, for binding to the peptide binding site on vancomycin.
This invention also provides the hybridoma cell line designated as HB 11834 and monoclonal antibodies produced thereby. Such monoclonal antibodies are most preferred for the quantification of vancomycin, most preferably by fluorescence polarization.
The invention also provides stable calibrators for use in quantifying vancomycin. The calibrators of the present invention are in an aqueous solution and contain a polypeptide stabilized vancomycin molecule. The polypeptide does not interfere with the binding of an antibody to the vancomycin molecule.
Also provided are kits useful for the quantification of vancomycin in a test sample having antibody reagents and labeled reagents. Preferred kits have antibodies produced from an immunogen of FIG. 6; most preferred are monoclonal IgG antibodies produced from an immunogen of FIG. 5.
The present invention also provides synthetic procedures for preparing immunogens which are employed for the production of such antibody reagents, and for preparing such labeled reagents.